Rhizobial Resource Associated with Epidemic Legumes in Tibet

A total of 128 bacterial test strains originated from Astragalus, Caragana, Gueldenstaedtia, Medicago, Melilotus, Oxytropis, Trifolium, and Vicia grown in Tibet were characterized phenotypically and genomically. Based upon the consensus of grouping results, they were identified as 16 putative species. Twenty-five test strains belonging to seven putative species of Agrobacterium, Bradyrhizobium, and Rhizobium might be nonsymbiotic bacteria and the remaining 103 test strains were symbiotic bacteria belonging to Mesorhizobium, Rhizobium, and Sinorhizobium meliloti. Although no novel taxon was detected in the symbiotic bacteria, several characters including the alkaliphilic psychrotolerance revealed that the Tibetan rhizobia could be ecotypes adapted to the local conditions. The results also demonstrated that frequent lateral transfer of symbiotic genes might have happened in the Tibetan rhizobia since nodC genes similar to that of S. meliloti were found in several Rhizobium test strains and all the Mesorhizobium species had very similar nodC genes despite their genomic background. All of these findings demonstrated that the Tibetan rhizobia were an important resource for further studies on rhizobial ecology and application. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor Cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.     

Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho‐GDIα, Rac1, and p38 pathway

Cyclic strain is an important inducer of proliferation and migration of vascular smooth muscle cells (VSMCs) which are involved in vascular remodeling during hypertension. However, its mechanism remains to be elucidated. VSMCs of rat aorta were exposed to cyclic strains in vitro with defined parameters, the static, 5%‐strain (physiological) and 15%‐strain (pathological), at 1.25 Hz for 24 h respectively. Then the possible signaling molecules participated in strain‐induced VSMC migration and proliferation were investigated. The results showed that 15%‐strain significantly increased VSMC migration and proliferation in comparison with 5%‐strain. Expression of Rho GDP dissociation inhibitor alpha (Rho‐GDIα) was repressed by 15%‐strain, but expressions of phospho‐Rac1 and phospho‐p38 were increased. Expressions of phospho‐Akt and phospho‐ERK1/2 were similar between the static, 5%‐strain and 15%‐strain groups. Rho‐GDIα “knock‐down” by target siRNA transfection increased migration and proliferation of VSMCs, and up‐regulated phosphorylation of Rac1 and p38 in all groups. Rac1 “knock‐down” repressed migration and proliferation of VSMCs, down‐regulated phosphorylation of p38, but had no effect on Rho‐GDIα expression. When siRNAs of Rho‐GDIα and Rac1 were co‐transfected to VSMCs, the expressions of Rho‐GDIα and phospho‐Rac1 were both decreased, and the effects of Rho‐GDIα “knock‐down” were blocked. Rho‐GDIα “knock‐down” promoted while Rac1 “knock‐down” postponed the assembly of stress fibers and focal adhesions in static. The results demonstrate that the pathological cyclic strain might induce migration and proliferation of VSMCs via repressing expression of Rho‐GDIα, which subsequently verified phosphorylations of Rac1 and p38. J. Cell. Biochem. 109: 906–914, 2010. © 2010 Wiley‐Liss, Inc.     

Application of organic solvent system for lipase-catalyzed regioselective benzoylation of 1-<Emphasis Type="Italic">β</Emphasis>-D-arabinofuranosylcytosine

In this paper, enzymatic regioselective acylation of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl benzoate (VB) using immobilized Candida antarctica lipase B in binary organic solvents was explored. It was found that the lipase showed high regioselectivity (> 99%) towards the 5′-OH of ara-C in the representative organic solvent mixture (hexane-pyridine). To understand the enzymatic processes and provide a fair comparison of hexane-pyridine with C₄MIm·PF₆-pyridine (the representative ionic liquid-containing system), the effect of each process variable on the reactions in hexane-pyridine was investigated. The results indicate that the optimum hexane content, initial a _w , molar ratio of VB to ara-C, and temperature were 28% (v/v), 0.11, 15, and 40°C, respectively. Under optimized conditions, the initial reaction rate in hexanepyridine (44.4 mM/h) was much higher than that in C₄MIm·PF₆-pyridine (29.4 mM/h) for each case. The maximum conversion yield, however, was increased when the reaction system was shifted from hexane-pyridine to C₄MIm·PF₆-pyridine. Further study revealed that the presence of an acidic by-product (benzoate acid, released during the acylation process) may cause rapid inactivation of the enzyme in hexane-pyridine, leading to a lower conversion rate, whereas the ionic liquid may have coating and protecting effects on the lipase during the reaction.     

Characterization of a new SCCmec element in Staphylococcus cohnii

Background

Many SCCmec elements of coagulase-negative staphylococci (CoNS) could not be typed using multiplex PCR. Such a ‘non-typable’ SCCmec was encountered in a Staphylococcus cohnii isolate.

Methodology/Principal Findings

The SCCmec type of methicillin-resistant S. cohnii clinical isolate WC28 could not be assigned using multiplex PCR. Newly-designed primers were used to amplify ccrA and ccrB genes. The whole SCCmec was obtained by three overlapping long-range PCR, targeting regions from left-hand inverted repeat (IRL) to ccrA/B, from ccrA/B to mecA and from mecA to orfX. The region abutting IRL was identified using inverse PCR with self-ligated enzyme-restricted WC28 fragments as the template. WC28 SCCmec had a class A mec gene complex (mecI-mecR1-mecA). The ccrA and ccrB genes were closest (89.7% identity) to ccrA _SHP of Staphylococcus haemolyticus strain H9 and to ccrB3 (90% identity) of Staphylococcus pseudintermedius strain KM241, respectively. Two new genes potentially encoding AAA-type ATPase were found in J1 region and a ψTn554 transposon was present in J2 region, while J3 region was the same as many SCCmec of Staphylococcus aureus. WC28 SCCmec abutted an incomplete SCC element with a novel allotype of ccrC, which was closest (82% identity) to ccrC1 allele 9 in Staphylococcus saprophyticus strain ATCC 15305. Only two direct target repeat sequences, one close to the 3′-end of orfX and the other abutting the left end of WC28 SCCmec, could be detected.

Conclusions/Significance

A new 35-kb SCCmec was characterized in a S. cohnii isolate, carrying a class A mec gene complex, new variants of ccrA5 and ccrB3 and two novel genes in the J1 region. This element is flanked by 8-bp perfect inverted repeats and is similar to type III SCCmec in S. aureus and a SCCmec in S. pseudintermedius but with different J1 and J3 regions. WC28 SCCmec was arranged in tandem with an additional SCC element with ccrC, SCC_WC28, but the two elements might have integrated independently rather than constituted a composite. This study adds new evidence of the diversity of SCCmec in CoNS and highlights the need for characterizing the ‘non-typable’ SCCmec to reveal the gene pool associated with mecA.     

Low level of genetic diversity in cultivated Pigeonpea compared to its wild relatives is revealed by diversity arrays technology

Understanding the distribution of genetic diversity among individuals, populations and gene pools is crucial for the efficient management of germplasm collections and breeding programs. Diversity analysis is routinely carried out using sequencing of selected gene(s) or molecular marker technologies. Here we report on the development of Diversity Arrays Technology (DArT) for pigeonpea (Cajanus cajan) and its wild relatives. DArT tests thousands of genomic loci for polymorphism and provides the binary scores for hundreds of markers in a single hybridization-based assay. We tested eight complexity reduction methods using various combinations of restriction enzymes and selected PstI/HaeIII genomic representation with the largest frequency of polymorphic clones (19.8%) to produce genotyping arrays. The performance of the PstI/HaeIII array was evaluated by typing 96 accessions representing nearly 20 species of Cajanus. A total of nearly 700 markers were identified with the average call rate of 96.0% and the scoring reproducibility of 99.7%. DArT markers revealed genetic relationships among the accessions consistent with the available information and systematic classification. Most of the diversity was among the wild relatives of pigeonpea or between the wild species and the cultivated C. cajan. Only 64 markers were polymorphic among the cultivated accessions. Such narrow genetic base is likely to represent a serious impediment to breeding progress in pigeonpea. Our study shows that DArT can be effectively applied in molecular systematics and biodiversity studies.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Use of an ionic liquid to improve asymmetric reduction of 4′-methoxyacetophenone catalyzed by immobilized Rhodotorula sp. AS2.2241 cells

Rhodotorula sp. AS2.2241, a newly isolated strain, was used as biocatalyst for asymmetric reduction of 4′-methoxyacetophenone (MOAP) to enantiopure (S)-1-(4-methoxyphenyl)ethanol {(S)-MOPE}. Despite the improved efficiency of the reaction with immobilized cells compared to free cells, the inhibition of the reaction by substrate and product in monophasic aqueous system proved to be big problem. For high efficient biotransformation, several water-immiscible ionic liquids (ILs) were employed as green solvents to construct ionic liquid-involving biphasic systems. Of the six ILs tested, C₄MIM·PF₆ exhibited the best biocompatibility with the cells, and consequently the biocatalytic reduction proceeded with the fastest initial reaction rate and the highest maximum substrate conversion in the C₄MIM·PF₆-based biphasic system. To better understand the bioreduction conducted in the C₄MIM·PF₆-based biphasic system, various variables that influenced the performance of the reaction were examined. The optimal buffer pH, reaction temperature, volume ratio of buffer to C₄MIM·PF₆ and substrate concentration were 7.5, 25 °C, 4/1 and 40 mM, respectively. Under the optimal conditions, the initial reaction rate, maximum substrate conversion and product e.e. were 1.6 μmol/h, 95.5% and >99%, respectively. Additionally, the cells still remained above 90% of their original activity in the C₄MIM·PF₆-based biphasic system, which was much higher than that in the monophasic buffer system (about 25% of their original activity), after being repeatedly used for 8 batches (50 h per batch), indicating that C₄MIM·PF₆ markedly enhanced the operational stability of the cells.     

MAMMALIAN REMAINS FROM THE PLIOCENE OF THE HANSHUI RIVER BASIN, SHAANXI

<正> A lot of mammalian remains from Yangjiawan village, the Hanshui River basin, Mianxian, Shaanxi, were collected by a field team of IVPP and the Geological Museum of Shaanxi Province in the autumn of 1984. Fossil mammals found in Yangjiawan Formation include 13 species (including three new species) belonging to 12 genera. This paper will give a preliminary study of the mammalian remains and observation of stratigraphic sections of several localities.     

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of ¹²⁵I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronection receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.